The goal of this study is to develop strategies to reduce/eliminate inhibitory antibodies in hemophilia A patients following repeated factor VIII infusion or gene therapy. Formation of inhibitory antibodies is a major problem in protein replacement therapy and expected to be a problem in gene therapy for hemophilia A patients. Approximately 25-50% of hemophilic patients develop anti-FVIII antibodies after repeated infusion of recombinant or pooled hFVIII protein. Following successful transfer of the factor VIII gene into the livers of hemophilia A mice by hydrodynamic-based nonviral gene transfer of a liver-specific, high-expressing FVIII plasmid, resulting in Supra-physiological levels of FVIII. We observed a robust immune response against FVIII two weeks post-treatment. This led to complete inhibition of circulating FVIII activity. This inhibitory response was mediated by a Th2-dominant human immune response. This inhibitory response completely ablated FVIII activity despite continued (essentially lifelong) FVIII production. This animal model is an ideal system for developing strategies to ameliorate the immune responses against FVIII. Recent data have demonstrated a critical role that CD4+CD25* regulatory T (Treg) cells in the regulation and suppression of autoimmune and alloimmune responses. Clinical trials of Treg infusion in BMT patients to prevent graft-versus-host disease (GVHD) are under way. Furthermore, Foxp3 which encodes the transcription factor Scurfin, is a master regulatory gene for the development and function of CD4+CD25+ regulatory T cells. In mice, ex vivo retroviral gene transfer of Foxp3 can convert peripheral CD25-CD45RO-CD4+ naive T cells into a regulatory T cell phenotype similar to CD4+CD25+ regulatory T cells. In addition, Treg type 1 cells (Tr1 cells) which can be induced by specific antigen and IL-10 have also been shown to maintain peripheral tolerance. We anticipate that an effective method to produce FVIII-specific CD4+CD25+ Treg cells (and/or Tr1) cells could provide a novel strategy to eliminate existing inhibitory antibodies and potentially induce long-term tolerance for hemophilia A patients with inhibitors. In this proposal we will test the hypotheses that: 1) Overexpression of Foxp3 in hemophilia A mice will modulate the immune responses against FVIII following gene transfer; 2) Lentiviral-based Foxp3 expression within FVIII-specific CD4+ T cells will induce CD4+CD25+Foxp3+ Treg cells. Further we predict that transplantation of such FVIII-specific Treg cells into hemophilia A mice will induce tolerance against FVIII; and 3) Lentiviral-based Foxp3 expression within FVIII-specific CD4+ T cells isolated from human hemophilia A patients with inhibitors can be used to generate FVIII-specific CD4+CD25+Foxp3+ Treg cells. This cell population could potentially be utilized in the future for cellular therapy in this disease setting.